The present invention relates to apparatus and methods for carrying out multiple different operations on multiple articles and establishing the operations on each article by its position in the array.
The present invention relates to apparatus and method useful in creating combinatorial chemistry libraries. More particularly, the present invention relates to apparatus and methods for synthesizing spatially-dispersed positionally encoded combinatorial chemistry libraries of oligomer whereby the synthesis is carried out on a plurality of solid supports which in turn are distributed in the form of a series of arrays. The position of each solid support in each array determines the exact identity of the oligomer.
The screening of chemical libraries to identify compounds which have novel pharmacological and material science properties is a common practice. These chemical libraries may be a collection of structurally related oligopeptides, oligonucleotides, small or large molecular weight organic or inorganic molecules. Those practiced in the art of combinatorial chemistry can accomplish the synthesis of combinatorial chemical libraries using two general methods. These methods are known to those skilled in the art as xe2x80x9cspatially-addressablexe2x80x9d methods and xe2x80x9csplit-poolxe2x80x9d methods. It is common to practice these methods using solid support chemical synthesis techniques as discussed by Gordon, et al.
A common feature to the spatially-addressable combinatorial library methods is that a unique combination of monomers is reacted to form a single oligomer or compound or, alternately, set of oligomer or compounds at a predefined unique physical location or address in the synthesis process. An example of the spatially-addressable method is provided by Geysen et al. and involves the generation of peptide libraries on an array of immobilized polymeric pins (a solid support) that fit the dimensions of a 96-well microtiter plate. A two-dimensional matrix of combinations is generated in each microtiter plate experiment, where nxc3x97m unique oligomer or compounds are produced for a combination of n+rn parallel monomer addition steps. The structure of each of the individual library members is determined by analyzing the pin location and the monomers employed at that address during the sequence of reaction steps in the synthesis.
An advantage of this method is that individual oligomer or compound products can be released from the polymeric pin surface in a spatially-addressable manner to allow isolation and screening of each discrete member of the library. Another advantage of this method is that the number of solid supports required is equal to, i.e. no larger than, the number of library members to be synthesized. Thus, relatively large quantities, i.e. micromolar quantities, of individual library members are synthesized in a practical manner using this method.
Related to the Geysen pin method are the parallel synthesis methods which use a reaction vessel system such as that practiced by Cody, et al. This is the practice of distributing a quantity of solid supports, such as chemically-derivatized polymeric resin beads (namely those of the composition polystyrene, polystyrene grafted with polyethylene glycol, or polyacrylimide, etc.) in a two dimensional matrix of nxc3x97m individual reaction vessels allowing the parallel addition of a set of nxc3x97m reactive monomers to produce a set of nxc3x97m oligomer; or compounds. This spatially-addressable method has advantages similar to that of Geysen, et al. Thus, individual oligomer or compound products can be released from the solid support in a spatially-addressable manner to allow isolation and screening of each discrete member of the library. Additionally, the number of solid supports required is equal to, i.e. no larger than, the number of library members to be synthesized. Thus, relatively large quantities, i.e. micromolar to millimolar quantities, of individual library members also are synthesized in a practical manner using this method.
Another example of a spatially-addressable method is the photo lithographic method for synthesizing a collection oligomer or compounds on the chemically-derivatized surface of a chip (a solid support) provided by Fodor et al. A variety of masking strategies can be employed to selectively remove photochemically-labile protecting groups thus revealing reactive functional groups at defined spatial locations on the chip. The functional groups are reacted with a monomer by exposing the chip surface to appropriate reagents. The sequential masking and reaction steps are recorded, thus producing a pre-defined record of discrete oligomer or compounds at known spatial addresses in an experiment. An advantage of this method is that binary masking strategies can be employed to produce a unique oligomer or compounds for n masking and monomer addition cycles. Two important disadvantages of this method are that a) relatively minute quantities are produced on the surface of the chip and; b) release and isolation of individual library members is not technically feasible.
Split-pool combinatorial library methods differ from spatially addressable methods in that the physical location of each unique oligomer or compound is not discrete. Instead, pools of library members are manipulated throughout the experiment. There are two major categories of split-pool methods currently in practice. These are: 1) deconvolution method S7 pioneered by Furka et al. and Houghten, et al. and 2) encoded methods by Gallop et al., Still, et al. and others.
It is common in the practice to employ solid support-based chemistry for these methods. A collection of solid supports are split into individual pools. These pools are then exposed to a series of reactive monomers, followed by a recombination step, in which the position of all solid supports is randomized. The solid supports are then split into a new set of individual pools, exposed to a new series of reactive monomers, followed by a second recombination step. By repeating this split, react and recombine process all possible combinations of oligomer or compounds from the series of monomers employed are obtained, providing a large excess of solid supports are utilized.
The number of oligomer or compounds obtained in an experiment is equal to the product of the monomers employed, however, the number of chemical transformation steps required is only equal to the sum of the monomers employed. Therefore, a geometric amplification of oligomer or compounds is realized relative to the amount of chemical transformation steps employed. For instance, only nine (9) transformation steps were employed using three (3) amino acid monomers in a three step process for the combinatorial synthesis of 27 peptide oligomer.
The prior art split-pool methods produce pools of oligomer or compounds as a product of the experiment. Therefore, the identification of a specific member of the library is typically found by screening the pools for a desired activity, biological or otherwise. The disadvantages of the deconvolution split-pool methods are that (a) the technique always requires that large mixtures of oligomer are screened in bioassays, (b) sequential rounds of resynthesis and bioassay are always required to deconvolute a library, and (c) since single oligomer are not produced a library is always stored as a mixture, requiring later deconvolution.
In the practice of encoded split-pool methods physical separation of the solid support is required to accomplish two tasks: first, to physically isolate the individual library member after screening and, second, to de-code the identity of the tag and thus deduce the chemical structure of the member. A disadvantage specific to the chemically encoded split-pool methods is that chemical tags introduce potential side reactions and failures both with orthogonal linkers and with tags, thus requiring compatibility between the tag chemistry and the chemistry utilized to synthesize the combinatorial library.
In practice, both categories of split-pool methods require a large excess of solid support beads to ensure with reasonable certainty (99% confidence level) that all possible oligomer are made when a random split-pool strategy is employed. This is necessary because the exact identity of each bead (i.e. the identity of each oligomer) is lost due to the unstructured nature of the split-pool method. This presents a significant problem when scaling up these methods for the production of micromole or larger amounts of individual oligomer in the library.
The parent application discloses a technique in the combinatorial chemistry art which can achieve geometric amplification in the number of library members synthesized relative to the number of synthetic steps required but, additionally, (a) avoids the need for chemical encoding steps (b) produces micromolar or larger amounts of individual oligomer; (c) uses only the number of solid supports required for the number of possible oligomer in the library; and (d) produces the oligomer in spatially-dispersed arrays wherein the identity of the oligomer is determined by its location in the array.
There is a need in the combinatorial chemistry art for apparatus which can carry out the processes and methods of the parent application.
There is a need in the combinatorial chemistry art for apparatus which can carry out the processes and methods of producing the oligomer in spatially-dispersed arrays wherein the identity of the oligomer is determined by its location in the array.
Glossary
Monomer: As used herein, a xe2x80x9cmonomerxe2x80x9d is any atom or molecule capable of forming at least one chemical bond. Thus, a xe2x80x9cmonomerxe2x80x9d is any member of the set of atoms or molecules that can be joined together as single units in a multiple of sequential or concerted chemical or enzymatic reaction steps to form an oligomer. Monomers may have one or a plurality of functional groups, which functional groups may be, but need not be, identical. The set of monomers useful in the present invention includes, but is not restricted to, alkyl and aryl amines; alkyl and aryl mercaptans; alkyl and aryl ketones; alkyl and aryl carboxylic acids; alkyl and aryl esters; alkyl and aryl ethers; alkyl and aryl sulfoxides; alkyl and aryl sulfones; alkyl and aryl sulfonamides; phenols; alkyl alcohols; alkyl and aryl alkenes; alkyl and aryl lactams; alkyl and aryl lactones; alkyl and aryl di- and polyenes; alkyl and aryl alkynes; alkyl and aryl unsaturated ketones; alkyl and aryl aldehydes; heteroatomic compounds containing one or more of the atoms of nitrogen, sulfur, phosphorous, oxygen, and other polyfunctional molecules containing one or more of the above functional groups; L-amino acids; D-amino acids; deoxyribonucleosides; deoxyribonucleotides; ribonucleosides; ribonucleotides; sugars; benzodiazepines; P-lactams; hydantoins; quinones; hydroquinones; terpenes; and the like. The monomers of the present invention may have groups protecting the functional groups within the monomer. Suitable protecting groups will depend on the functionality and particular chemistry used to construct the library. Examples of suitable functional protecting groups will be readily apparent to skilled artisans, and are described, for example, in Greene and WUtS,14 which is incorporated herein by reference. As used herein, xe2x80x9cmonomerxe2x80x9d refers to any member of a basis set for synthesis of an oligomer.
For example, the dimers of 20 L-amino acids form a basis set of 400 xe2x80x9cmonomersxe2x80x9d for synthesis of polypeptides. Different basis sets of monomers may be used at successive steps in the synthesis of an oligomer.
Oligomer: As used herein, an xe2x80x9coligomerxe2x80x9d is any chemical structure that can be synthesized using the combinatorial library methods and apparatus of this invention, including, for example, amides, esters, thiGethers, ketones, ethers, sulfoxides, sulfonamides, sulfones, phosphates, alcohols, aldehydes, alkenes, alkynes, aromatics, polyaromatics, heterocyclic compounds containing one or more of the atoms of nitrogen, sulfur, oxygen, and phosphorous, and the like; chemical entities having a common core structure such as, for example, terpenes, steroids, P-lactams, benzodiazepines, xanthates, indoles, indolones, lactones, lactams, hydantoins, quiriones, hydroquinones, and the like; chains of repeating monomer units such as polysaccharides, phospholipids, polyurethanes, polyesters, polycarbonates, poly ureas, polyamides, polyethyleneimines, poly arylene sulfides, polyimides, polyacetates, polypeptides, polynucleotides, and the like; or other oligomer as will be readily apparent to one skilled in the art upon review of this disclosure. Thus, an xe2x80x9coligomerxe2x80x9d of the present invention may be linear, branched, cyclic, or assume various other forms as will be apparent to those skilled in the art. Thus, xe2x80x9coligomerxe2x80x9d may be used synonymously or interchangeably with xe2x80x9ccompoundxe2x80x9d, thus describing any structure, organic or inorganic, which can be produced in a sequential fashion via the addition of monomeric units as described above.
Solid Support: A xe2x80x9csolid supportxe2x80x9d as used herein is a material, or combination of materials, having a rigid or semi-rigid surface and having functional groups or linkers, or that is capable of being chemically derivatized with functional groups or linkers, that are suitable for carrying out chemical synthesis reactions. Such materials will preferably take the form of small beads, pellets, disks, cylinders, capillaries, hollow fibers, needles, solid fibers, cellulose beads, pore-glass beads, silica gels, polystyrene beads cross-linked with divinylbenzene and optionally grafted with polyethylene glycol, grafted co-poly beads, poly-acrylamide beads, latex beads, dirnethylacrylamide beads optionally cross-linked with N,Nxe2x80x2-bis-hycryloyl ethylene diamine, polydimethylacrylainide beads crosslinked with polystyrene, glass particles coated with a hydrophobic polymer, or other convenient forms. xe2x80x9cSolid supportsxe2x80x9d may be constructed such that they are capable of being transferred mechanically from one support carrier to another support carrier.
Linker: A xe2x80x9clinkerxe2x80x9d is a moiety, molecule, or group of molecules attached to a solid support and spacing a synthesized oligomer from the solid support. Typically a linker will be bi-functional, wherein said linker has a functional group at one end capable of attaching to a monomer, oligomer, or solid support, a series of spacer residues, and a functional group at another end capable of attaching to a monomer, oligomer, or solid support. The functional groups may be, but need not be, identical. Additionally, said linker may be cleaved by a chemical transformation such that the synthesized oligomer, or part of the synthesized oligomer, or the synthesized oligomer and the linker, or the synthesized oligomer and part of the linker may be chemically separated from the solid support, linker, or both.
Carrier: A carrier as used herein is a portable support structure or platform which may be in the form of a tray, grid or other form for positionally holding s plurality of solid supports in predetermined spatial arrays. A carrier can take any number of forms suitable for receiving and temporarily holding solid supports in a desirable spatial array. A xe2x80x9cdonor carrierxe2x80x9d is a carrier that is loaded with solid supports and is in a position to transfer the solid supports to a donee carrier. A xe2x80x9cdonee or recipient carrierxe2x80x9d is an empty carrier that is readied or positioned to receive solid supports from a donor carrier.
Column: The term xe2x80x9ccolumnxe2x80x9d as used herein for the arrangement of the solid supports means a vertical row. That is, column means a row that extends toward and away from the observer or vertically from top toward the bottom of a page.
Row: The term row as used herein for the arrangement of the solid supports means a horizontal row that extends left to right on a page.
A primary object of the present invention is to provide an apparatus and method for the synthesis of a spatially-dispersed combinatorial library of oligomer, in which the oligomer are distributed in a controlled manner.
In accordance with a primary aspect of the invention, a system for moving multiple supports in parallel through multiple different synthesis steps to provide a library of oligomer comprises at least two monomers, a plurality of synthesis supports, a first plurality of support carriers wherein each carrier has a uniform array of distinct support holding positions for said synthesis supports, means for contacting each array of synthesis supports with a different monomer to provide a first chemical transformation of said synthesis supports, a second plurality of support carriers wherein each carrier has a uniform array of distinct support holding positions for receiving said chemically transformed synthesis supports contained in said first plurality of support carriers, transfer apparatus for transferring a selected row or column of synthesis supports from each of said first plurality of carriers to each of said second plurality of carriers to enable the supports in the second to undergo at least a second chemical transformation, and whereby each support position in each carrier identifies the chemical compound thereon.
An object of the present invention is to provide an improved apparatus and method for the synthesis of a spatially-dispersed combinatorial library of oligomer, in which the oligomer are distributed in a controlled manner. These oligomers are comprised of a series of monomers which are introduced into the oligomer in a sequential and stepwise fashion via chemical transformation steps (hereafter referred to as xe2x80x9cstepsxe2x80x9d). These monomers are comprised of subsets of monomers such that the first subset of monomers is introduced in the first step, the second set of monomers is introduced in the second step, etc. The method further comprises apparatus for holding a plurality of solid supports in a sequence of spatial arrays during steps of chemical transformation steps and moving them in a sequential and stepwise fashion between transformation steps. The method comprises means for introducing the monomers in a sequential and stepwise fashion on a series of solid supports. The number of supports equals the number of oligomer in the library.
A novel aspect of this apparatus and process as distinguished from the prior art is that the supports are arranged in, and subsequently redistributed in a controlled manner between, a series of arrays. This series of arrays are enabled by means for holding the supports in physically discrete locations such that the exact identity of each support is provided by its location. The series of arrays of supports are placed in a further series of reaction vessels for the individual steps of an oligomer synthesis. After each step in the oligomer synthesis the supports are redistributed in a predetermined controlled manner from one series of arrays to a next series of arrays.
A further novel aspect of this process is that between each step the redistribution of the supports is carried out in a controlled fashion, such that all possible combinations of possible oligomer are synthesized. A further novel aspect of this process is that the positions of all supports are known during the synthesis experiment such that the identity of an oligomer is unequivocally established by its physical location. Thus, the applied method achieves a geometric amplification in the number of library members synthesized relative to the number of synthetic steps required while providing individual library members in a spatially dispersed format. Thus, the use of a tagging system is eliminated for a split-pool synthesis experiment.
The apparatus and method has utility in the production of oligomer which are available for screening in assays for novel biological, chemical, or physical properties which may have commercial value. Further, the structures of these oligomer are readily identifiable by virtue of their physical location. The apparatus further provides a means for producing each oligomer in a discrete physical location which allows any pre-determined oligomer to be readily isolated from all other oligomer in the library. Yet another advantage of the invention is that no excess of solid supports is required, thus enabling a larger scale of synthesis.